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18 results on '"MESH : Base Sequence"'

1. Binding of the Methyl Donor S -Adenosyl- <scp>l</scp> -Methionine to Middle East Respiratory Syndrome Coronavirus 2′- O -Methyltransferase nsp16 Promotes Recruitment of the Allosteric Activator nsp10

Author

Wahiba Aouadi, Bruno Canard, Alexandre Blanjoie, Jean-Jacques Vasseur, Françoise Debart, Etienne Decroly, Architecture et fonction des macromolécules biologiques (AFMB), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut National de la Recherche Agronomique (INRA), Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Architecture et fonction des macromolécules biologiques ( AFMB ), Centre National de la Recherche Scientifique ( CNRS ) -Aix Marseille Université ( AMU ) -Institut National de la Recherche Agronomique ( INRA ), Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] ( IBMM ), Ecole Nationale Supérieure de Chimie de Montpellier ( ENSCM ) -Université de Montpellier ( UM ) -Centre National de la Recherche Scientifique ( CNRS ), and Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, S-Adenosylmethionine, Endoglycosidase, Adenosine, Methyltransferase, Viral Nonstructural Proteins, MESH: Base Sequence, MESH : Protein Multimerization, MESH: Allosteric Regulation, [SDV.IMM.II]Life Sciences [q-bio]/Immunology/Innate immunity, [ SDV.BBM.BC ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Bacteroides, MESH : Viral Nonstructural Proteins, Phylogeny, MESH : Methyltransferases, GacS, chemistry.chemical_classification, MESH: Kinetics, biology, MESH: Protein Multimerization, MESH : Protein Binding, Methylation, MESH : S-Adenosylmethionine, MESH : Allosteric Regulation, Genome Replication and Regulation of Viral Gene Expression, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], 3. Good health, Biochemistry, MESH: RNA, Viral, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Middle East Respiratory Syndrome Coronavirus, RNA, Viral, MESH : Kinetics, Protein Binding, MESH : Adenosine, RNA virus, Immunology, Allosteric regulation, MESH : Middle East Respiratory Syndrome Coronavirus, [ SDV.MP.VIR ] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Microbiology, MESH: Methylation, Two-component system, 03 medical and health sciences, Allosteric Regulation, Pseudomonas, Virology, MESH: Methyltransferases, MESH : RNA, Viral, biochemistry, MESH: Protein Binding, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Binding site, [ SDV.IMM.II ] Life Sciences [q-bio]/Immunology/Innate immunity, Base Sequence, Protein, MESH: Middle East Respiratory Syndrome Coronavirus, Viral translation, MESH: S-Adenosylmethionine, RNA, Methyltransferases, MESH : Methylation, Glycoside hydrolase 39, MESH: Adenosine, biology.organism_classification, NMR, Kinetics, 030104 developmental biology, Enzyme, RNA processing, chemistry, Insect Science, MESH: Viral Nonstructural Proteins, MESH : Base Sequence, Protein Multimerization
Abstract

The Middle East respiratory syndrome coronavirus (MERS-CoV) nonstructural protein 16 (nsp16) is an S -adenosyl- l -methionine (SAM)-dependent 2′- O -methyltransferase (2′-O-MTase) that is thought to methylate the ribose 2′-OH of the first transcribed nucleotide (N 1 ) of viral RNA cap structures. This 2′-O-MTase activity is regulated by nsp10. The 2′- O methylation prevents virus detection by cell innate immunity mechanisms and viral translation inhibition by the interferon-stimulated IFIT-1 protein. To unravel the regulation of nsp10/nsp16 2′-O-MTase activity, we used purified MERS-CoV nsp16 and nsp10. First, we showed that nsp16 recruited N7-methylated capped RNA and SAM. The SAM binding promotes the assembly of the enzymatically active nsp10/nsp16 complex that converted 7m GpppG (cap-0) into 7m GpppG 2′Om (cap-1) RNA by 2′-OH methylation of N 1 in a SAM-dependent manner. The subsequent release of SAH speeds up nsp10/nsp16 dissociation that stimulates the reaction turnover. Alanine mutagenesis and RNA binding assays allowed the identification of the nsp16 residues involved in RNA recognition forming the RNA binding groove (K46, K170, E203, D133, R38, Y47, and Y181) and the cap-0 binding site (Y30, Y132, and H174). Finally, we found that nsp10/nsp16 2′-O-MTase activity is sensitive to known MTase inhibitors, such as sinefungin and cap analogues. This characterization of the MERS-CoV 2′-O-MTase is a preliminary step toward the development of molecules to inhibit cap 2′-O methylation and to restore the host antiviral response. IMPORTANCE MERS-CoV codes for a cap 2′- O -methyltransferase that converts cap-0 into cap-1 structure in order to prevent virus detection by cell innate immunity mechanisms. We report the biochemical properties of MERS-CoV 2′O-methyltransferase, which is stimulated by nsp10 acting as an allosteric activator of the nsp16 2′- O -methyltransferase possibly through enhanced RNA binding affinity. In addition, we show that SAM promotes the formation of the active nsp10/nsp16 complex. Conversely, after cap methylation, the reaction turnover is speeded up by cap-1 RNA release and nsp10/nsp16 complex dissociation, at the low intracellular SAH concentration. These results suggest that SAM/SAH balance is a regulator of the 2′- O -methyltransferase activity and raises the possibility that SAH hydrolase inhibitors might interfere with CoV replication cycle. The enzymatic and RNA binding assays developed in this work were also used to identify nsp16 residues involved in cap-0 RNA recognition and to understand the action mode of known methyltransferase inhibitors.

Published
2017

2. Characterization of Unique Signature Sequences in the Divergent Maternal Protein Bcl2l10

Author

Germain Gillet, Abdel Aouacheria, François Penin, Aurélie Cornut-Thibaut, Yannis Guillemin, Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche en Cancérologie de Lyon (UNICANCER/CRCL), Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de Biologie Moléculaire de la Cellule (LBMC), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Ganivet, Agnès, Institut de biologie et chimie des protéines [Lyon] ( IBCP ), Université Claude Bernard Lyon 1 ( UCBL ), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique ( CNRS ), Centre de Recherche en Cancérologie de Lyon ( CRCL ), Université de Lyon-Université de Lyon-Centre Léon Bérard [Lyon]-Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Laboratoire de Biologie Moléculaire de la Cellule ( LBMC ), École normale supérieure - Lyon ( ENS Lyon ) -Université Claude Bernard Lyon 1 ( UCBL ), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), and École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL)

Subjects
MESH : Cell Line, MESH : Molecular Sequence Data, Amino Acid Motifs, MESH: Amino Acid Sequence, MESH: Base Sequence, MESH : Proto-Oncogene Proteins c-bcl-2, [ SDV.CAN ] Life Sciences [q-bio]/Cancer, MESH: INDEL Mutation, protein sequence, MESH: Amino Acid Motifs, MESH: Protein Structure, Tertiary, 0302 clinical medicine, INDEL Mutation, Databases, Genetic, genetic polymorphism, MESH : Female, MESH: Animals, MESH: Genetic Variation, [SDV.BDD]Life Sciences [q-bio]/Development Biology, Peptide sequence, MESH: Databases, Genetic, MESH: Evolution, Molecular, Sequence Deletion, Genetics, chemistry.chemical_classification, 0303 health sciences, MESH : Amino Acid Sequence, MESH : Sequence Alignment, apoptosis, Vertebrate, MESH: Sequence Deletion, Amino acid, Proto-Oncogene Proteins c-bcl-2, MESH: Calcium, 030220 oncology & carcinogenesis, Female, MESH : Protein Structure, Tertiary, MESH : Amino Acid Motifs, Molecular Sequence Data, MESH: Sequence Alignment, [SDV.CAN]Life Sciences [q-bio]/Cancer, Sequence alignment, Biology, Cell Line, MESH: Oocytes, Evolution, Molecular, 03 medical and health sciences, Bcl-2 family, MESH : Genetic Variation, biology.animal, evolution, [SDV.BDD] Life Sciences [q-bio]/Development Biology, Genetic variation, Homologous chromosome, Animals, Humans, MESH : HeLa Cells, MESH : Evolution, Molecular, Amino Acid Sequence, MESH : Calcium, MESH : Databases, Genetic, Indel, MESH : INDEL Mutation, Molecular Biology, Ecology, Evolution, Behavior and Systematics, 030304 developmental biology, MESH : Mutagenesis, Insertional, calcium, MESH: Humans, MESH: Molecular Sequence Data, Base Sequence, MESH : Sequence Deletion, MESH: Apoptosis, MESH : Humans, MESH : Oocytes, Genetic Variation, Protein Structure, Tertiary, MESH: Cell Line, Mutagenesis, Insertional, MESH: Mutagenesis, Insertional, MESH: Proto-Oncogene Proteins c-bcl-2, chemistry, MESH: HeLa Cells, Oocytes, MESH : Base Sequence, MESH : Animals, Sequence Alignment, MESH: Female, MESH : Apoptosis, HeLa Cells
Abstract

International audience; Insertions or deletions (indels) of amino acids residues have been recognized as an important source of genetic and structural divergence between paralogous Bcl-2 family members. However, these signature sequences have not so far been extensively investigated amongst orthologous Bcl-2 family proteins. Bcl2l10 is an antiapoptotic member of the Bcl-2 family that has evolved rapidly throughout the vertebrate lineage and which shows conserved abundant expression in eggs and oocytes. In this paper, we have unraveled two major sites of divergence between human Bcl2l10 and its vertebrate homologs. The first one provides length variation at the N-terminus (before the BH4 domain) and the second one is located between the predicted α5-α6 pore-forming helices, providing an unprecedented case in the superfamily of helix-bundled pore-forming proteins. These two particular indels were studied phylogenetically and through biochemical and cell biological techniques, including truncation and site-directed mutagenesis. While deletion of the N-terminal extension had no significant functional impact in HeLa cells, our results suggest that the human Bcl2l10 protein evolved a calcium-binding motif in its α5-α6 interhelical region by acquiring critical negatively charged residues. Considering the reliance of female eggs on calcium-dependent proteins and calcium-regulated processes and the exceptional longevity of oocytes in the primate lineage, we propose that this microstructural variation may be an adaptive feature associated with high maternal expression of this Bcl-2 family member.

Published
2011

3. Somatic mutations activating STAT3 in human inflammatory hepatocellular adenomas. : Activating STAT3 mutations in hepatocellular adenoma

Author

Jeanne Tran Van Nhieu, Michel Bihl, Karine Poussin, Mohamed Amessou, Paulette Bioulac-Sage, Jessica Zucman-Rossi, Charles Balabaud, Camilla Pilati, Jean-Charles Nault, Tina Izard, Valérie Paradis, Gabrielle Couchy, Genomique Fonctionnelle des Tumeurs Solides, Université Paris Diderot - Paris 7 ( UPD7 ) -IFR105-Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), Labex Immuno-oncology, Centre de Recherche des Cordeliers ( CRC ), Université Pierre et Marie Curie - Paris 6 ( UPMC ) -École pratique des hautes études ( EPHE ) -Université Paris Diderot - Paris 7 ( UPD7 ) -Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Université Pierre et Marie Curie - Paris 6 ( UPMC ) -École pratique des hautes études ( EPHE ) -Université Paris Diderot - Paris 7 ( UPD7 ) -Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -PRES Sorbonne Paris Cité-Faculté de Médecine, Fibrose hépatique et cancer du foie, Université Bordeaux Segalen - Bordeaux 2-IFR66-Institut National de la Santé et de la Recherche Médicale ( INSERM ), Département de pathologie [Mondor], Assistance publique - Hôpitaux de Paris (AP-HP)-Hôpital Henri Mondor-Université Paris-Est Créteil Val-de-Marne - Paris 12 ( UPEC UP12 ), Service de Pathologie [Clichy], Assistance publique - Hôpitaux de Paris (AP-HP)-Hôpital Beaujon, Department of Cancer Biology, The Scripps Research Institute, Service de pathologie [Bordeaux], Université Bordeaux Segalen - Bordeaux 2-CHU Bordeaux [Bordeaux]-Groupe hospitalier Pellegrin, Service d'Hépato-Gastro Entérologie et Oncologie Digestive, Assistance publique - Hôpitaux de Paris (AP-HP)-Hôpital Européen Georges Pompidou [APHP] ( HEGP ), and This work was supported by the association pour la recherche Contre le Cancer (ARC, grant n°3194), Inserm (Réseaux de recherche clinique et réseaux de recherche en santé des populations), Collection Nationale des Carcinomes Hépatocellulaires, the Ligue Nationale Contre le Cancer ('Cartes d'identité des tumeurs' program) and the BioIntelligence collaborative program. C.P., M.A. and J-C.N. are supported by a fellowship from the MENRT, Inca and ARC, respectively. T.I. is supported by the National Institutes of Health grants GM071596, AI055894, and AI067949.

Subjects
MESH : Tyrosine, Male, Models, Molecular, MESH : Liver Neoplasms, MESH : Mutant Proteins, MESH : Aged, MESH : RNA, Small Interfering, SH2 domain, medicine.disease_cause, HNF1A, STAT3, MESH : Phosphorylation, 0302 clinical medicine, MESH : STAT3 Transcription Factor, Cytokine Receptor gp130, Immunology and Allergy, CTNNB1, MESH : Female, SOCS3, Phosphorylation, RNA, Small Interfering, 0303 health sciences, Mutation, Liver Neoplasms, DNA, Neoplasm, MESH : Adult, Middle Aged, SAA, 3. Good health, MESH : Cytokine Receptor gp130, JAK1, src-Family Kinases, JAK2, MESH : Dimerization, 030220 oncology & carcinogenesis, [ SDV.MHEP.HEG ] Life Sciences [q-bio]/Human health and pathology/Hépatology and Gastroenterology, Female, MESH : DNA, Neoplasm, MESH : Mutation, CRP, IL6ST, Dimerization, Tyrosine kinase, Src, Signal Transduction, Proto-oncogene tyrosine-protein kinase Src, MESH : Carcinoma, Hepatocellular, Adult, STAT3 Transcription Factor, Carcinoma, Hepatocellular, MESH : Interleukin-6, MESH : Models, Molecular, MESH : Male, Immunology, Active Transport, Cell Nucleus, Biology, IFN, Adenoma, Liver Cell, gp130, 03 medical and health sciences, MESH : Adenoma, Liver Cell, MESH : Protein Structure, Quaternary, Cell Line, Tumor, medicine, Humans, MESH : Middle Aged, Protein Structure, Quaternary, MESH : Active Transport, Cell Nucleus, Aged, 030304 developmental biology, MESH : Signal Transduction, IL-6, Base Sequence, MESH : Cell Line, Tumor, Interleukin-6, MESH : Humans, Brief Definitive Report, beta-catenin, digestive system diseases, STAT protein, Inflammatory Hepatocellular Adenoma, Cancer research, Tyrosine, MESH : src-Family Kinases, MESH : Base Sequence, Mutant Proteins, Carcinogenesis
Abstract

Somatic STAT3 mutations present in a subset of inflammatory hepatocellular adenomas result in the generation of constitutively active STAT3 proteins that homodimerize independently of IL-6 stimulation., Inflammatory hepatocellular adenomas (IHCAs) are benign liver tumors. 60% of these tumors have IL-6 signal transducer (IL6ST; gp130) mutations that activate interleukin 6 (IL-6) signaling. Here, we report that 12% of IHCA subsets lacking IL6ST mutations harbor somatic signal transducer and activator of transcription 3 (STAT3) mutations (6/49). Most of these mutations are amino acid substitutions in the SH2 domain that directs STAT3 dimerization. In contrast to wild-type STAT3, IHCA STAT3 mutants constitutively activated the IL-6 signaling pathway independent of ligand in hepatocellular cells. Indeed, the IHCA STAT3 Y640 mutant homodimerized independent of IL-6 and was hypersensitive to IL-6 stimulation. This was associated with phosphorylation of tyrosine 705, a residue required for IL-6–induced STAT3 activation. Silencing or inhibiting the tyrosine kinases JAK1 or Src, which phosphorylate STAT3, impaired constitutive activity of IHCA STAT3 mutants in hepatocellular cells. Thus, we identified for the first time somatic STAT3 mutations in human tumors, revealing a new mechanism of recurrent STAT3 activation and underscoring the role of the IL-6–STAT3 pathway in benign hepatocellular tumorigenesis.

Published
2011

4. Aberrant DNA methylation distinguishes hepatocellular carcinoma associated with HBV and HCV infection and alcohol intake

Author

Fabien Zoulim, Marie-Pierre Lambert, Massimo Tommasino, Zdenko Herceg, Jörg Tost, Jean-Yves Scoazec, Anupam Paliwal, Pierre Hainaut, Thomas Vaissière, Bakary S. Sylla, Isabelle Chemin, Centre de Recherche en Cancérologie de Lyon (CRCL), Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Interactions Bactéries-Cellules (UIBC), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris]-Institut National de la Recherche Agronomique (INRA), International Agency for Cancer Research (IACR), Equipe 16, Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 (UCBL), Service d'hépatologie et de gastroentérologie, Hospices Civils de Lyon (HCL)-Hôtel-Dieu-Hospices Civils de Lyon (HCL)-Hôtel-Dieu-Centre de Recherche en Cancérologie de Lyon (CRCL), Centre International de Recherche contre le Cancer - International Agency for Research on Cancer (CIRC - IARC), Organisation Mondiale de la Santé / World Health Organization Office (OMS / WHO), Sect Mech Carcinogenesis, equipe 4, Laboratoire Central d'Anatomie et de Cytologie Pathologiques [Hôpital Edouard Herriot - HCL], Hôpital Edouard Herriot [CHU - HCL], Hospices Civils de Lyon (HCL)-Hospices Civils de Lyon (HCL)-Hôpital Edouard Herriot [CHU - HCL], Hospices Civils de Lyon (HCL)-Hospices Civils de Lyon (HCL)-Centre de Recherche en Cancérologie de Lyon (CRCL), Centre National de Génotypage (CNG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Centre de Recherche en Cancérologie de Lyon ( CRCL ), Université Claude Bernard Lyon 1 ( UCBL ), Université de Lyon-Université de Lyon-Centre Léon Bérard [Lyon]-Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Interactions Bactéries-Cellules ( UIBC ), Institut National de la Recherche Agronomique ( INRA ) -Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale ( INSERM ), International Agency for Cancer Research ( IACR ), International Agency for Cancer Research, Université de Lyon-Université de Lyon-Centre Léon Bérard [Lyon]-Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ) -Université Claude Bernard Lyon 1 ( UCBL ), Hospices Civils de Lyon ( HCL ) -Hôtel-Dieu-Hospices Civils de Lyon ( HCL ) -Hôtel-Dieu-Centre de Recherche en Cancérologie de Lyon ( CRCL ), Centre International de Recherche contre le Cancer - International Agency for Research on Cancer ( CIRC - IARC ), Organisation mondiale de la santé (OMS/WHO), international Agency for Research on Cancer - IARC, Hospices Civils de Lyon ( HCL ) -Hospices Civils de Lyon ( HCL ) -Hôpital Edouard Herriot [CHU - HCL], Hospices Civils de Lyon ( HCL ) -Hospices Civils de Lyon ( HCL ) -Centre de Recherche en Cancérologie de Lyon ( CRCL ), Centre National de Génotypage ( CNG ), Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ), Centre de Recherche en Cancérologie de Lyon (UNICANCER/CRCL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 (UCBL), Hospices Civils de Lyon (HCL)-Hôtel-Dieu-Hospices Civils de Lyon (HCL)-Hôtel-Dieu-Centre de Recherche en Cancérologie de Lyon (UNICANCER/CRCL), Hospices Civils de Lyon (HCL)-Hospices Civils de Lyon (HCL)-Centre de Recherche en Cancérologie de Lyon (UNICANCER/CRCL), and Institut National de la Recherche Agronomique (INRA)-Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
Male, MESH : Liver Neoplasms, MESH : Aged, Receptors, Nicotinic, MESH: Base Sequence, medicine.disease_cause, [ SDV.CAN ] Life Sciences [q-bio]/Cancer, MESH: DNA Methylation, 0302 clinical medicine, Risk Factors, MESH: Liver Neoplasms, MESH: Risk Factors, MESH : Female, MESH: Gene Silencing, MESH: Carcinoma, Hepatocellular, MESH: Glutathione S-Transferase pi, MESH: Aged, 0303 health sciences, MESH: Middle Aged, MESH : Tumor Suppressor Proteins, Liver Neoplasms, RNA-Binding Proteins, DNA, Neoplasm, Hepatitis C, Methylation, Middle Aged, MESH : Adult, Hepatitis B, MESH : Risk Factors, 3. Good health, DNA-Binding Proteins, 030220 oncology & carcinogenesis, Hepatocellular carcinoma, DNA methylation, MESH: Receptors, Nicotinic, Female, MESH : DNA Primers, MESH : DNA, Neoplasm, MESH : DNA-Binding Proteins, Adult, MESH : Carcinoma, Hepatocellular, MESH: DNA Primers, Carcinoma, Hepatocellular, Alcohol Drinking, MESH : Male, Hepatitis C virus, MESH : RNA-Binding Proteins, MESH: DNA, Neoplasm, [SDV.CAN]Life Sciences [q-bio]/Cancer, Biology, MESH: Phosphoproteins, 03 medical and health sciences, MESH : Gene Silencing, medicine, Humans, MESH : Middle Aged, MESH: Tumor Suppressor Proteins, Gene Silencing, neoplasms, Aged, DNA Primers, 030304 developmental biology, MESH: Hepatitis C, Hepatitis B virus, MESH: Humans, Base Sequence, MESH : Receptors, Nicotinic, MESH: Hepatitis B, Hepatology, Tumor Suppressor Proteins, MESH : Humans, MESH : Hepatitis B, Cancer, MESH: Adult, DNA Methylation, MESH : Hepatitis C, Phosphoproteins, medicine.disease, MESH: Male, digestive system diseases, MESH : Alcohol Drinking, MESH: RNA-Binding Proteins, Glutathione S-Transferase pi, MESH : Glutathione S-Transferase pi, Immunology, MESH : Base Sequence, MESH : Phosphoproteins, MESH : DNA Methylation, MESH: Female, MESH: Alcohol Drinking, MESH: DNA-Binding Proteins
Abstract

International audience; BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is one of the most frequent human cancers and a major cause of cancer-related death worldwide. The major risk factors for developing HCC are infection by hepatitis B virus (HBV) and hepatitis C virus (HCV), chronic alcoholism, and aflatoxins; however, critical gene targets remain largely unknown. Herein, we sought to establish DNA methylation patterns in HCC and corresponding cirrhotic tissues and to identify DNA methylation changes associated with major risk factors. METHODS: We have established assays for quantitative analysis of DNA methylation levels in a panel of seven cancer-associated genes and repetitive elements, and combined these assays with a series of HCC tumors, associated with major risk factors, collected from two different geographical areas. RESULTS: We found a high frequency of aberrant hypermethylation of specific genes (RASSF1A, GSTP1, CHRNA3, and DOK1) in HCC tumors as compared to control cirrhotic or normal liver tissues, suggesting that aberrant hypermethylation exhibits non-random and tumor-specific patterns in HCC. Importantly, our analysis revealed an association between alcohol intake and the hypomethylation of MGMT and between hypermethylation of GSTP1 and HBV infection, indicating that hypermethylation of the genes analyzed in HCC tumors exhibits remarkably distinct patterns depending on associated risk factors. CONCLUSIONS: This study identifies aberrant DNA methylation of specific cellular genes in HCC and the major risk factors associated with these changes, providing information that could be exploited for biomarker discovery in clinics and molecular epidemiology.

Published
2011

5. Murine APOBEC1 Is a Powerful Mutator of Retroviral and Cellular RNA In Vitro and In Vivo

Author

Denise Guetard, Marc Sitbon, Jean-Pierre Vartanian, Anne Keriel, Myrtille Renard, Simon Wain-Hobson, Vincent Petit, Rétrovirologie Moléculaire, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut de Génétique Moléculaire de Montpellier (IGMM), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique ( CNRS ), Institut de Génétique Moléculaire de Montpellier ( IGMM ), and Université de Montpellier ( UM ) -Centre National de la Recherche Scientifique ( CNRS )

Subjects
Hypoxanthine Phosphoribosyltransferase, MESH : Molecular Sequence Data, MESH : RNA, Messenger, MESH : DNA, Complementary, MESH : NIH 3T3 Cells, viruses, Muscle Proteins, MESH: Base Sequence, Nucleic Acid Denaturation, MESH : Retroviridae Infections, MESH: Animals, Newborn, Mice, chemistry.chemical_compound, MESH : Hypoxanthine Phosphoribosyltransferase, Structural Biology, Murine leukemia virus, MESH : RNA, MESH: Animals, APOBEC Deaminases, MESH : Cytidine Deaminase, Gammaretrovirus, MESH : Muscle Proteins, 0303 health sciences, biology, Nucleotides, MESH : Animals, Newborn, 030302 biochemistry & molecular biology, MESH: Leukemia Virus, Murine, Cytidine, Cytidine deaminase, MESH: Apolipoproteins B, 3. Good health, Leukemia Virus, Murine, MESH: Retroviridae Infections, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, MESH : Mutation, MESH: Genome, Viral, MESH : Genome, Viral, MESH : Leukemia Virus, Murine, MESH : Nucleic Acid Denaturation, APOBEC, DNA, Complementary, MESH: Mutation, APOBEC-1 Deaminase, Molecular Sequence Data, MESH: Leukemia, Experimental, Genome, Viral, MESH: Nucleic Acid Denaturation, [ SDV.MP.VIR ] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, MESH: Muscle Proteins, 03 medical and health sciences, MESH: RNA, Cytidine Deaminase, MESH : Mice, MESH : RNA Editing, Animals, Humans, MESH: RNA Editing, RNA, Messenger, MESH: Mice, Molecular Biology, Apolipoproteins B, MESH: RNA, Messenger, MESH: Cytidine Deaminase, 030304 developmental biology, Messenger RNA, Leukemia, Experimental, MESH: Molecular Sequence Data, MESH: Humans, Base Sequence, APOBEC1, MESH : Humans, MESH: Tumor Virus Infections, RNA, MESH : Apolipoproteins B, MESH: DNA, Complementary, biology.organism_classification, MESH: Hypoxanthine Phosphoribosyltransferase, Molecular biology, MESH: Nucleotides, Tumor Virus Infections, Animals, Newborn, chemistry, Mutation, NIH 3T3 Cells, MESH : Nucleotides, MESH : Leukemia, Experimental, MESH : Base Sequence, MESH : Animals, RNA Editing, MESH : Tumor Virus Infections, Retroviridae Infections, MESH: NIH 3T3 Cells
Abstract

International audience; Mammalian APOBEC molecules comprise a large family of cytidine deaminases with specificity for RNA and single-stranded DNA (ssDNA). APOBEC1s are invariably highly specific and edit a single residue in a cellular mRNA, while the cellular targets for APOBEC3s are not clearly established, although they may curtail the transposition of some retrotransposons. Two of the seven member human APOBEC3 enzymes strongly restrict human immunodeficiency virus type 1 in vitro and in vivo. We show here that ssDNA hyperediting of an infectious exogenous gammaretrovirus, the Friend-murine leukemia virus, by murine APOBEC1 and APOBEC3 deaminases occurs in vitro. Murine APOBEC1 was able to hyperdeaminate cytidine residues in murine leukemia virus genomic RNA as well. Analysis of the edited sites shows that the deamination in vivo was due to mouse APOBEC1 rather than APOBEC3. Furthermore, murine APOBEC1 is able to hyperedit its primary substrate in vivo, the apolipoprotein B mRNA, and a variety of heterologous RNAs. In short, murine APOBEC1 is a hypermutator of both RNA and ssDNA in vivo, which could exert occasional side effects upon overexpression.

Published
2009

6. Primary cystic lung light chain deposition disease: a clinicopathologic entity derived from unmutated B cells with a stereotyped IGHV4-34/IGKV1 receptor

Author

Marc Stern, Marie-Hélène Delfau-Larue, Hervé Mal, Diane Damotte, Magali Colombat, Michel Fournier, Patrice Callard, Jacques Diebold, Jean-Pierre Farcet, Christiane Copie-Bergman, Service d'anatomie pathologique [CHU Tenon], Assistance publique - Hôpitaux de Paris (AP-HP)-CHU Tenon [APHP], Service de pneumologie B, Assistance publique - Hôpitaux de Paris (AP-HP)-AP-HP - Hôpital Bichat - Claude Bernard [Paris]-Université Paris Diderot - Paris 7 ( UPD7 ), Service d'anatomie pathologique, Assistance publique - Hôpitaux de Paris (AP-HP)-Hôtel-Dieu, Département de pathologie [Mondor], Assistance publique - Hôpitaux de Paris (AP-HP)-Hôpital Henri Mondor-Université Paris-Est Créteil Val-de-Marne - Paris 12 ( UPEC UP12 ), Service d'anatomo-pathologie [CHU HEGP], Assistance publique - Hôpitaux de Paris (AP-HP)-Hôpital Européen Georges Pompidou [APHP] ( HEGP ), Service d'immunologie biologique, Service de pneumologie, and Hôpital Foch [Suresnes]

Subjects
Lung Diseases, Pathology, MESH : Molecular Sequence Data, MESH : DNA, medicine.medical_treatment, Immunoglobulin Variable Region, Paraproteinemias, MESH : Immunoglobulin Heavy Chains, Plasma cell, Biochemistry, 0302 clinical medicine, MESH : Female, MESH : Immunoglobulin Variable Region, B-Lymphocytes, 0303 health sciences, MESH : Lung Diseases, Cysts, MESH : Amino Acid Sequence, Hematology, MESH : Adult, MESH : Immunoglobulin Light Chains, 3. Good health, medicine.anatomical_structure, MESH : Paraproteinemias, 030220 oncology & carcinogenesis, MESH : Lung Transplantation, Female, Genes, Immunoglobulin Light Chain, Immunoglobulin Heavy Chains, Lung Transplantation, Adult, medicine.medical_specialty, Genes, Immunoglobulin Heavy Chain, Molecular Sequence Data, Immunology, MESH : Genes, Immunoglobulin Heavy Chain, MESH : Cysts, Biology, Immunoglobulin light chain, Article, Light chain deposition disease, MESH : B-Lymphocytes, Lung Disorder, Immunoglobulin kappa-Chains, 03 medical and health sciences, MESH : Immunoglobulin kappa-Chains, medicine, Humans, Lung transplantation, Amino Acid Sequence, [ SDV.BBM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology, MESH : Genes, Immunoglobulin Light Chain, 030304 developmental biology, Lung, Base Sequence, MESH : Humans, DNA, Cell Biology, medicine.disease, Immunoglobulin heavy chain, MESH : Base Sequence, Immunoglobulin Light Chains, CD5
Abstract

We have recently described a new form of light chain deposition disease (LCDD) presenting as a severe cystic lung disorder requiring lung transplantation. There was no bone marrow plasma cell proliferation. Because of the absence of disease recurrence after bilateral lung transplantation and of serum-free light chain ratio normalization after the procedure, we hypothesized that monoclonal light chain synthesis occurred within the lung. The aim of this study was to look for the monoclonal B-cell component in 3 patients with cystic lung LCDD. Histologic examination of the explanted lungs showed diffuse nonamyloid κ light chain deposits associated with a mild lymphoid infiltrate composed of aggregates of small CD20+, CD5−, CD10− B lymphocytes reminiscent of bronchus-associated lymphoid tissue. Using polymerase chain reaction (PCR), we identified a dominant B-cell clone in the lung in the 3 studied patients. The clonal expansion of each patient shared an unmutated antigen receptor variable region sequence characterized by the use of IGHV4-34 and IGKV1 subgroups with heavy and light chain CDR3 sequences of more than 80% amino acid identity, a feature evocative of an antigen-driven process. Combined with clinical and biologic data, our results strongly argue for a new antigen-driven primary pulmonary lymphoproliferative disorder.

Published
2008

7. A unique conformation of the anticodon stem-loop is associated with the capacity of tRNAfMet to initiate protein synthesis

Author

Carine Tisné, Yves Mechulam, Frédéric Dardel, Pierre Barraud, Emmanuelle Schmitt, Laboratoire de cristallographie et RMN biologiques ( LCRB - UMR 8015 ), Université Paris Descartes - Paris 5 ( UPD5 ) -Centre National de la Recherche Scientifique ( CNRS ), Laboratoire de Biochimie de l'Ecole polytechnique ( BIOC ), École polytechnique ( X ) -Centre National de la Recherche Scientifique ( CNRS ), Laboratoire de cristallographie et RMN biologiques (LCRB - UMR 8015), Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biochimie de l'Ecole polytechnique (BIOC), École polytechnique (X)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5), and Centre National de la Recherche Scientifique (CNRS)-École polytechnique (X)

Subjects
Models, Molecular, MESH : Escherichia coli, MESH : Molecular Sequence Data, MESH : Nucleic Acid Conformation, MESH: Base Sequence, Crystallography, X-Ray, chemistry.chemical_compound, MESH : Anticodon, Protein biosynthesis, MESH : RNA, Transfer, Met, Peptide Chain Initiation, Translational, 0303 health sciences, MESH: Escherichia coli, 030302 biochemistry & molecular biology, Stem-loop, RNA, Bacterial, MESH: Nucleic Acid Conformation, Biochemistry, Transfer RNA, MESH: RNA, Bacterial, MESH: Models, Molecular, MESH: Peptide Chain Initiation, Translational, RNA, Transfer, Met, MESH : Models, Molecular, Base pair, Stereochemistry, Molecular Sequence Data, Biology, 03 medical and health sciences, Eukaryotic translation, Anticodon, Escherichia coli, MESH: Anticodon, Genetics, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [ SDV.BBM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology, 030304 developmental biology, MESH: Molecular Sequence Data, Methionine, Base Sequence, MESH: RNA, Transfer, Met, RNA, MESH : RNA, Bacterial, MESH: Crystallography, X-Ray, chemistry, Helix, Nucleic Acid Conformation, MESH : Base Sequence, MESH : Crystallography, X-Ray, MESH : Peptide Chain Initiation, Translational
Abstract

International audience; In all organisms, translational initiation takes place on the small ribosomal subunit and two classes of methionine tRNA are present. The initiator is used exclusively for initiation of protein synthesis while the elongator is used for inserting methionine internally in the nascent polypeptide chain. The crystal structure of Escherichia coli initiator tRNA(f)(Met) has been solved at 3.1 A resolution. The anticodon region is well-defined and reveals a unique structure, which has not been described in any other tRNA. It encompasses a Cm32*A38 base pair with a peculiar geometry extending the anticodon helix, a base triple between A37 and the G29-C41 pair in the major groove of the anticodon stem and a modified stacking organization of the anticodon loop. This conformation is associated with the three GC basepairs in the anticodon stem, characteristic of initiator tRNAs and suggests a mechanism by which the translation initiation machinery could discriminate the initiator tRNA from all other tRNAs.

Published
2008

8. Genetic diversity of Echinococcus multilocularis on a local scale

Author

Jenny Knapp, Bruno Gottstein, Patrick Giraudoux, M-H Guislain, Francis Raoul, Jean-Mathieu Bart, Renaud Piarroux, Laboratoire Chrono-environnement ( LCE ), Université Bourgogne Franche-Comté ( UBFC ) -Centre National de la Recherche Scientifique ( CNRS ) -Université de Franche-Comté ( UFC ), Institute of Parasitology, Laboratoire Chrono-environnement - CNRS - UBFC (UMR 6249) (LCE), Centre National de la Recherche Scientifique (CNRS)-Université de Franche-Comté (UFC), and Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université Bourgogne Franche-Comté [COMUE] (UBFC)

Subjects
MESH : Molecular Sequence Data, Range (biology), Foxes, MESH: Base Sequence, MESH : Echinococcus multilocularis, MESH : Microsatellite Repeats, MESH: Variation (Genetics), 0302 clinical medicine, MESH : Genes, Helminth, Parasite hosting, MESH: Animals, MESH : Female, 610 Medicine & health, MESH: Phylogeny, Genes, Helminth, Phylogeny, [SDV.EE]Life Sciences [q-bio]/Ecology, environment, 0303 health sciences, 630 Agriculture, MESH : Variation (Genetics), Ecology, Infectious Diseases, Microsatellite, Female, France, Microbiology (medical), Molecular Sequence Data, 030231 tropical medicine, Zoology, Biology, Echinococcus multilocularis, MESH: Sequence Homology, Nucleic Acid, Microbiology, [ SDV.EE ] Life Sciences [q-bio]/Ecology, environment, 03 medical and health sciences, Phylogenetics, Sequence Homology, Nucleic Acid, parasitic diseases, Genetic variation, Genetics, Animals, Helminths, MESH : Sequence Homology, Nucleic Acid, MESH : France, Molecular Biology, Ecology, Evolution, Behavior and Systematics, 030304 developmental biology, MESH: Echinococcus multilocularis, MESH : Foxes, Genetic diversity, MESH: Foxes, MESH: Molecular Sequence Data, Base Sequence, Genetic Variation, MESH : Phylogeny, biology.organism_classification, MESH: France, MESH : Base Sequence, MESH: Microsatellite Repeats, MESH : Animals, MESH: Female, Microsatellite Repeats, MESH: Genes, Helminth
Abstract

International audience; Echinococcus multilocularis is the causative agent of human Alveolar Echinococcosis (AE), and it is one of the most lethal zoonotic infections in the Northern Hemisphere. In France, the eastern and central regions are endemic areas; Franche-Comt?Lorraine and Auvergne are particularly contaminated. Recently, several human cases were recorded in the French Ardennes area, a region adjacent to the western border of the E. multilocularis range in France. A previous study in this focus described a prevalence of over 50% of the parasite in red foxes. The present study investigated the genetic diversity of adult worms collected from foxes in a 900km(2) area in the Ardennes. Instead of a conventional mitochondrial target (ATP6), two microsatellite targets (EmsB and NAK1) were used. A total of 140 adult worms isolated from 25 red foxes were genotyped. After hierarchical clustering analyses, the EmsB target enabled us to distinguish two main assemblages, each divided into sub-groups, yielding the differentiation of six clusters or assemblage profiles. Thirteen foxes (52% of the foxes) each harbored worms from at least two different assemblage profiles, suggesting they had become infected by several sources. Using the NAK1 target, we identified 3 alleles, two found in association with the two EmsB assemblages. With the NAK1 target, we investigated the parasite breeding system and the possible causes of genetic diversification. Only one fox harbored hybrid worms, indicative of a possible (and rare) occurrence of recombination, although multiple infections have been observed in foxes. These results confirm the usefulness of microsatellite targets for assessing genetic polymorphism in a geographically restricted local range.

Published
2008

9. Hypoxia Down-regulates CCAAT/Enhancer Binding Protein-α Expression in Breast Cancer Cells

Author

Robert Barouki, Etienne Blanc, Nathalie M. Mazure, Fabrice Lecuru, Marie-Claude Fulchignoni-Lataud, Marie-Aude Le Frere Belda, Anne Dreiem, Thérèse Hervèe Mayi, Charbel Massaad, Vincent Favaudon, Liliane Massaad-Massade, Ramzi Seifeddine, Laboratoire de Détection et de Géophysique (CEA) (LDG), DAM Île-de-France (DAM/DIF), Direction des Applications Militaires (DAM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction des Applications Militaires (DAM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), IFR50, Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Faculté de Médecine Nice, Institut de signalisation, biologie du développement et cancer (ISBDC), Centre National de la Recherche Scientifique (CNRS)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA), Génotoxicologie, signalisation et radiothérapie expérimentale, Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Biophysique moléculaire, Régulation de la transcription et maladies génétiques (RTMG), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Stéroïdes et système nerveux : physiopathologie moléculaire et clinique, Institut National de la Santé et de la Recherche Médicale (INSERM), Faculté de Médecine Paris-Sud, Université Paris-Sud - Paris 11 (UP11), Toxicologie Moleculaire (U490), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), Laboratoire de Détection et de Géophysique (CEA) ( LDG ), Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ), Hôpital Européen Georges Pompidou [APHP] ( HEGP ), Université Nice Sophia Antipolis ( UNS ), Université Côte d'Azur ( UCA ) -Université Côte d'Azur ( UCA ) -Faculté de Médecine Nice, Institut de signalisation, biologie du développement et cancer ( ISBDC ), Université Côte d'Azur ( UCA ) -Université Côte d'Azur ( UCA ) -Centre National de la Recherche Scientifique ( CNRS ), Institut National de la Santé et de la Recherche Médicale ( INSERM ) -INSTITUT CURIE, Régulation de la transcription et maladies génétiques ( RTMG ), Centre National de la Recherche Scientifique ( CNRS ) -Centre National de la Recherche Scientifique ( CNRS ), Institut National de la Santé et de la Recherche Médicale ( INSERM ), Université Paris-Sud - Paris 11 ( UP11 ), Toxicologie Moleculaire, Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-Faculté de Médecine Nice, Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Curie

Subjects
Cancer Research, Hypoxia-Inducible Factor 1, MESH : Molecular Sequence Data, MESH : RNA, Messenger, Transcription, Genetic, MESH : Immunohistochemistry, MESH: Cell Hypoxia, Electrophoretic Mobility Shift Assay, MESH: Cell Cycle, RNA polymerase II, MESH : Blotting, Western, MESH : Breast Neoplasms, MESH: Base Sequence, MESH : RNA, Small Interfering, MESH: Down-Regulation, MESH : Down-Regulation, 0302 clinical medicine, Transcription (biology), MESH: Reverse Transcriptase Polymerase Chain Reaction, Enhancer binding, MESH: RNA, Small Interfering, Gene expression, MESH: CCAAT-Enhancer-Binding Protein-alpha, MESH : CCAAT-Enhancer-Binding Protein-alpha, RNA, Small Interfering, Promoter Regions, Genetic, 0303 health sciences, Gene knockdown, Reverse Transcriptase Polymerase Chain Reaction, Cell Cycle, MESH : Reverse Transcriptase Polymerase Chain Reaction, Immunohistochemistry, Cell Hypoxia, 3. Good health, MESH: Promoter Regions (Genetics), Oncology, MESH : Electrophoretic Mobility Shift Assay, 030220 oncology & carcinogenesis, MESH: Cell Growth Processes, MESH : Cell Hypoxia, MESH : Transfection, Subcellular Fractions, medicine.medical_specialty, MESH : Hypoxia-Inducible Factor 1, alpha Subunit, MESH: Cell Line, Tumor, Blotting, Western, Molecular Sequence Data, MESH : Deferoxamine, Down-Regulation, Breast Neoplasms, Cell Growth Processes, MESH : Subcellular Fractions, Deferoxamine, Biology, Transfection, MESH: Hypoxia-Inducible Factor 1, alpha Subunit, 03 medical and health sciences, Cell Line, Tumor, Internal medicine, MESH : Cell Cycle, CCAAT-Enhancer-Binding Protein-alpha, medicine, Humans, MESH: Blotting, Western, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, RNA, Messenger, [ SDV.BBM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Transcription factor, MESH: RNA, Messenger, 030304 developmental biology, MESH: Humans, MESH: Molecular Sequence Data, Base Sequence, MESH : Cell Line, Tumor, MESH: Transcription, Genetic, MESH: Transfection, MESH : Humans, MESH : Transcription, Genetic, MESH: Immunohistochemistry, MESH: Deferoxamine, Hypoxia-Inducible Factor 1, alpha Subunit, MESH : Promoter Regions (Genetics), Molecular biology, Endocrinology, HIF1A, MESH : Cell Growth Processes, MESH: Subcellular Fractions, MESH: Electrophoretic Mobility Shift Assay, biology.protein, MESH : Base Sequence, MESH: Breast Neoplasms
Abstract

International audience; The transcription factor CCAAT/enhancer binding protein-alpha (C/EBP alpha) is involved in the control of cell differentiation and proliferation, and has been suggested to act as a tumor suppressor in several cancers. By using microarray analysis, we have previously shown that hypoxia and estrogen down-regulate C/EBP alpha mRNA in T-47D breast cancer cells. Here, we have examined the mechanism by which the down-regulation by hypoxia takes place. Using the specific RNA polymerase II inhibitor 5,6-dichlorobenzimidazole-1-beta-D-ribofuranoside, the mRNA stability was analyzed under normoxia or hypoxia by quantitative reverse transcription-PCR. Hypoxia reduced the half-life of C/EBP alpha mRNA by approximately 30%. C/EBP alpha gene promoter studies indicated that hypoxia also repressed the transcription of the gene and identified a hypoxia-responsive element (-522; -527 bp), which binds to hypoxia-inducible factor (HIF)-1 alpha, as essential for down-regulation of C/EBP alpha transcription in hypoxia. Immunocytochemical analysis showed that C/EBP alpha was localized in the nucleus at 21% O(2), but was mostly cytoplasmic under 1% O(2). Knockdown of HIF-1 alpha by RNAi restored C/EBP alpha to normal levels under hypoxic conditions. Immunohistochemical studies of 10 tumor samples did not show any colocalization of C/EBP alpha and glucose transporter 1 (used as a marker for hypoxia). Taken together, these results show that hypoxia down-regulates C/EBP alpha expression in breast cancer cells by several mechanisms, including transcriptional and posttranscriptional effects. The down-regulation of C/EBP alpha in hypoxia is mediated by HIF-1.

Published
2008

10. Investigating Alternative RNA Splicing in Xenopus

Author

Serge Hardy, Agnès Méreau, Institut de Génétique et Développement de Rennes ( IGDR ), Université de Rennes 1 ( UR1 ), Université de Rennes ( UNIV-RENNES ) -Université de Rennes ( UNIV-RENNES ) -IFR140-Centre National de la Recherche Scientifique ( CNRS ), Institut de Génétique et Développement de Rennes (IGDR), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES), Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS), and Le Corre, Morgane

Subjects
Male, Oocyte, MESH : Sequence Analysis, RNA, Embryo, Nonmammalian, MESH : RNA, Messenger, MESH : Molecular Sequence Data, Xenopus, MESH: Morpholinos, MESH : Alternative Splicing, MESH : Morpholinos, Xenopus Proteins, MESH: Base Sequence, MESH: Microinjections, Splicing, Primary transcript, Chorionic Gonadotropin, Morpholinos, Xenopus laevis, 0302 clinical medicine, MESH : Embryo, Nonmammalian, Gene expression, MESH: Sequence Analysis, RNA, MESH : Xenopus laevis, MESH: Animals, MESH : Female, Regulatory Elements, Transcriptional, MESH: Xenopus Proteins, MESH: Organ Specificity, MESH : Chorionic Gonadotropin, 0303 health sciences, biology, MESH: Alternative Splicing, MESH : Xenopus Proteins, MESH: Reproductive Control Agents, Organ Specificity, Embryo, Gene Knockdown Techniques, RNA splicing, Proteome, Reproductive Control Agents, Female, Microinjections, MESH : Male, Molecular Sequence Data, Computational biology, MESH: Oocytes, MESH : Organ Specificity, MESH: Regulatory Elements, Transcriptional, 03 medical and health sciences, MESH: Chorionic Gonadotropin, MESH: Xenopus laevis, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, RNA, Messenger, MESH : Regulatory Elements, Transcriptional, [ SDV.BBM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology, MESH: RNA, Messenger, 030304 developmental biology, Messenger RNA, MESH: Molecular Sequence Data, Base Sequence, Sequence Analysis, RNA, Mechanism (biology), MESH : Oocytes, Alternative splicing, MESH: Embryo, Nonmammalian, biology.organism_classification, MESH: Gene Knockdown Techniques, MESH : Reproductive Control Agents, MESH: Male, MESH : Microinjections, Oocytes, RNA, MESH : Base Sequence, MESH : Gene Knockdown Techniques, MESH : Animals, MESH: Female, 030217 neurology & neurosurgery
Abstract

International audience; Alternative splicing, the process by which distinct mature mRNAs can be produced from a single primary transcript, is a key mechanism to increase the organism complexity. The generation of alternative splicing pattern is a means to expand the proteome diversity and also to control gene expression through the regulation of mRNA abundance. Alternative splicing is therefore particularly prevalent during development and accordingly numerous splicing events are regulated in a tissue or temporal manner. To study the roles of alternative splicing during developmental processes and decipher the molecular mechanisms that underlie temporal and spatial regulation, it is important to develop in vivo whole animal studies. In this chapter, we present the advantages of using the amphibian Xenopus as a fully in vivo model to study alternative splicing and we describe the experimental procedures that can be used with Xenopus laevis embryos and oocytes to define the cis-regulatory elements and identify the associated trans-acting factors.

Published
2012

11. PNPLA1 mutations cause autosomal recessive congenital ichthyosis in golden retriever dogs and humans

Author

Anais Grall, Céline Derbois, Matthieu Le Gallo, Ingrid Hausser, Laetitia Lagoutte, Sébastien Küry, Judith Fischer, Catherine André, Emmanuelle Bourrat, Emmanuel Bensignor, Sandrine Planchais, Franz P.W. Radner, Éric Guaguère, Francis Galibert, Rudolf Zechner, Susanne Grond, Jacques Fontaine, Robert Zimmermann, Gwang-Jin Kim, Mark Lathrop, Frédérique Degorce-Rubiales, Anne Thomas, Didier Pin, Christophe Hitte, Institut de Génétique et Développement de Rennes (IGDR), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Clinique Vétérinaire Saint Bernard, Institute of Molecular Biosciences, Karl-Franzens-Universität Graz, Service de dermatologie [Paris], Université Paris Diderot - Paris 7 (UPD7)-Groupe Hospitalier Saint Louis - Lariboisière - Fernand Widal [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Department of dermatology, University clinic Heidelberg, Electron Microscopy Core Facility, Universität Heidelberg [Heidelberg], Centre National de Génotypage (CNG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Génomique d'Evry (IG), Institut de Biologie François JACOB (JACOB), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Institute for Human Genetics, University Clinic Freiburg, Faculty for Biology, University of Freiburg [Freiburg], Laboratoire d'Anatomie Pathologique Vétérinaire du Sud-Ouest, Animal Genetics Laboratory, Antagene, Service d'ORL et de Chirurgie Cervicofaciale, Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Trousseau [APHP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Clinique Vétérinaire de la Boulais, Clinique vétérinaire, Clinique vétérinaire, Bruxelles, Interactions Cellules Environnement - UR (ICE), VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS), Centre d'Etude du Polymorphisme Humain (CEPH), Université Paris Diderot - Paris 7 (UPD7)-Institut Universitaire d'Hématologie (IUH), Université Paris Diderot - Paris 7 (UPD7)-Fondation Jean Dausset, Zentrum für Biosystemanalyse, Albert-Ludwigs-Universität Freiburg, This study was supported by CNRS, the European Commission (FP7-LUPA, GA-201370). R. Zechner and R. Zimmermann were supported by the FWF F30 SFB Lipotox, Z136 Wittgenstein, the GEN-AU project GOLD by the Austrian Ministry of Science and Research and FFG. I.H. was supported by the NIRK Network (German BMBF 01GM0904)., European Project: 201370,EC:FP7:HEALTH,FP7-HEALTH-2007-A,LUPA(2008), Institut de Génétique et Développement de Rennes ( IGDR ), Université de Rennes 1 ( UR1 ), Université de Rennes ( UNIV-RENNES ) -Université de Rennes ( UNIV-RENNES ) -Centre National de la Recherche Scientifique ( CNRS ) -Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Assistance publique - Hôpitaux de Paris (AP-HP)-Groupe Hospitalier Saint Louis - Lariboisière - Fernand Widal [Paris]-Université Paris Diderot - Paris 7 ( UPD7 ), Centre National de Génotypage ( CNG ), Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ), Institut de Génomique d'Evry ( IG ), Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay, Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -CHU Trousseau [APHP], Unité de Dermatologie, VetAgro Sup ( VAS ), Centre d'Etude du Polymorphisme Humain ( CEPH ), Université Paris Diderot - Paris 7 ( UPD7 ) -Institut Universitaire d'Hématologie ( IUH ), Université Paris Diderot - Paris 7 ( UPD7 ) -Fondation Jean Dausset, European Project : 201370,EC:FP7:HEALTH,FP7-HEALTH-2007-A,LUPA ( 2008 ), Université de Rennes (UR)-Centre National de la Recherche Scientifique (CNRS)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Université Paris Diderot - Paris 7 (UPD7)-Groupe Hospitalier Saint Louis - Lariboisière - Fernand Widal [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Universität Heidelberg [Heidelberg] = Heidelberg University, Université Paris-Saclay-Institut de Biologie François JACOB (JACOB), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), CHU Trousseau [APHP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), De Villemeur, Hervé, and Unravelling the molecular basis of common complex human disorders using the dog as a model system - LUPA - - EC:FP7:HEALTH2008-01-01 - 2012-06-30 - 201370 - VALID

Subjects
Male, MESH : Molecular Sequence Data, [SDV.GEN] Life Sciences [q-bio]/Genetics, MESH: Base Sequence, medicine.disease_cause, 0403 veterinary science, MESH: Dogs, MESH: INDEL Mutation, MESH: Lipase, MESH : Dogs, INDEL Mutation, Congenital ichthyosis, Missense mutation, MESH : Female, MESH: Animals, MESH: Codon, Nonsense, Cells, Cultured, Skin, Genetics, 0303 health sciences, Mutation, 04 agricultural and veterinary sciences, Lamellar ichthyosis, MESH : Adult, MESH : Lipase, MESH : Dermatologic Agents, MESH : Codon, Nonsense, MESH: Ichthyosis, Lamellar, Codon, Nonsense, Female, MESH : Genome-Wide Association Study, MESH: Cells, Cultured, Adult, 040301 veterinary sciences, MESH : Male, Nonsense mutation, Molecular Sequence Data, Mutation, Missense, ALOX12B, Golden Retriever, Genes, Recessive, MESH: Nitrendipine, Biology, 03 medical and health sciences, Dogs, MESH: Skin, MESH : Cells, Cultured, medicine, MESH : Skin, Animals, Humans, Indel, MESH : INDEL Mutation, MESH: Genes, Recessive, 030304 developmental biology, MESH: Mutation, Missense, [SDV.GEN]Life Sciences [q-bio]/Genetics, MESH: Humans, MESH: Molecular Sequence Data, Base Sequence, Nitrendipine, MESH : Humans, MESH : Ichthyosis, Lamellar, MESH : Genes, Recessive, MESH: Adult, Lipase, [SDV.MHEP.DERM] Life Sciences [q-bio]/Human health and pathology/Dermatology, medicine.disease, MESH : Nitrendipine, MESH: Male, MESH: Dermatologic Agents, MESH: Genome-Wide Association Study, MESH : Base Sequence, MESH : Animals, Dermatologic Agents, [ SDV.GEN ] Life Sciences [q-bio]/Genetics, [ SDV.MHEP.DERM ] Life Sciences [q-bio]/Human health and pathology/Dermatology, MESH: Female, MESH : Mutation, Missense, [SDV.MHEP.DERM]Life Sciences [q-bio]/Human health and pathology/Dermatology, Ichthyosis, Lamellar, Genome-Wide Association Study
Abstract

International audience; Ichthyoses comprise a heterogeneous group of genodermatoses characterized by abnormal desquamation over the whole body, for which the genetic causes of several human forms remain unknown. We used a spontaneous dog model in the golden retriever breed, which is affected by a lamellar ichthyosis resembling human autosomal recessive congenital ichthyoses (ARCI), to carry out a genome-wide association study. We identified a homozygous insertion-deletion (indel) mutation in PNPLA1 that leads to a premature stop codon in all affected golden retriever dogs. We subsequently found one missense and one nonsense mutation in the catalytic domain of human PNPLA1 in six individuals with ARCI from two families. Further experiments highlighted the importance of PNPLA1 in the formation of the epidermal lipid barrier. This study identifies a new gene involved in human ichthyoses and provides insights into the localization and function of this yet uncharacterized member of the PNPLA protein family.

Published
2011

12. Natural polymorphisms of HIV-1 CRF01_AE integrase coding region in ARV-naive individuals in Cambodia, Thailand and Vietnam : an ANRS AC12 working group study

Author

Martine Peeters, Eric Nerrienet, Tawee Donchai, Ahidjo Ayouba, Ton Tran, Janin Nouhin, Marie-Laure Chaix, Nicole Ngo-Giang-Huong, Sreymom Ken, Truong Xuan Lien, Khanh Thu Huynh Hoang, Jiraporn Kamkorn, Institut Pasteur du Cambodge, Réseau International des Instituts Pasteur (RIIP), Épidémiologie clinique, santé mère-enfant et VIH en Asie du Sud-Est (IRD_PHPT), Harvard University-Chiang Mai University (CMU), Institut Pasteur d'Ho Chi Minh Ville, VIH/SIDA et maladies associées, Université Montpellier 1 (UM1), Infections à Vih, Réservoirs, Pharmacologie des Antirétroviraux et Prévention de la Transmission Mère Enfant, Université Paris Descartes - Paris 5 (UPD5), Immunology and Infectious Diseases, Harvard School of Public Health, Harvard University [Cambridge]-Chiang Mai university (Thaïlande), Institut Pasteur du Cambodge-Réseau International des Instituts Pasteur ( RIIP ), Épidémiologie clinique, santé mère-enfant et VIH en Asie du Sud-Est ( IRD_PHPT ), Réseau International des Instituts Pasteur ( RIIP ) -Institut Pasteur d'Ho Chi Minh Ville, Université Montpellier 1 ( UM1 ), Université Paris Descartes - Paris 5 ( UPD5 ), and Harvard University [Cambridge]-Chiang Mai University (CMU)

Subjects
MESH: CD4 Lymphocyte Count, MESH : Polymorphism, Genetic, Integrase inhibitor, HIV Infections, HIV Integrase, Drug resistance, MESH: Base Sequence, Polymerase Chain Reaction, MESH: HIV-1, 0302 clinical medicine, 030212 general & internal medicine, MESH: Anti-HIV Agents, MESH : Polymerase Chain Reaction, MESH : Algorithms, Genetics, 0303 health sciences, MESH: HIV Infections, Thailand, MESH: Amino Acid Substitution, 3. Good health, Integrase, Infectious Diseases, Vietnam, Lentivirus, MESH : DNA Primers, Cambodia, Algorithms, MESH : HIV-1, medicine.drug, MESH: DNA Primers, Microbiology (medical), Anti-HIV Agents, MESH: Algorithms, Biology, South East Asia, Microbiology, Virus, 03 medical and health sciences, MESH : Amino Acid Substitution, Resistance mutations, [ SDV.MHEP ] Life Sciences [q-bio]/Human health and pathology, HIV-1 integrase, MESH: Polymorphism, Genetic, MESH : CD4 Lymphocyte Count, MESH : HIV Infections, Consensus sequence, medicine, Humans, MESH: Thailand, Molecular Biology, Ecology, Evolution, Behavior and Systematics, DNA Primers, 030304 developmental biology, Polymorphism, Genetic, MESH: Humans, Base Sequence, MESH : Cambodia, MESH : Anti-HIV Agents, MESH: Cambodia, MESH : Humans, MESH: Polymerase Chain Reaction, MESH : Vietnam, MESH : Thailand, biology.organism_classification, Raltegravir, Virology, CD4 Lymphocyte Count, Multiple drug resistance, Amino Acid Substitution, HIV-1, biology.protein, MESH : Base Sequence, MESH: Vietnam, MESH: HIV Integrase, Antiretroviral naive, MESH : HIV Integrase, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract

International audience; The HIV integrase enzyme is essential for the HIV life cycle as it mediates integration of HIV-1 proviral DNA into the infected cell's genome. Recently, the development of drugs capable of inhibiting integrase has provided major new options for HIV-infected, treatment-experienced patients with multidrug resistant virus, as well treatment-naïve patients. More than 40 amino acid substitutions within integrase have been described as associated mostly with resistance of HIV B-subtypes to currently available integrase inhibitors (INIs). We have analyzed the natural polymorphisms of the integrase coding region in 87 antiretroviral-naïve subjects (32 from Cambodia, 37 from Thailand and 18 from Vietnam) infected with CRF01_AE virus, the predominant HIV-1 strain circulating in Southeast Asia. The 864bp integrase coding region was sequenced using the ANRS consensus sequencing technique from plasma samples, and amino acid results were interpreted for drug resistance according to the ANRS (Updated July 2009, version 18) and Stanford algorithms (Version November 6, 2009). Alignment of the 87 amino acid sequences against the 2004 Los Alamos HIV-1 clade B consensus sequence showed that overall, 119 of 288 (41.3%) amino acid positions presented at least one polymorphism each. Substitutions found in >60% of study subjects occurred at: K14, A21, V31, S39, I72, T112, T124, T125, G134, I135, K136, D167, V201, L234 and S283. Also, new amino acid substitutions of as yet unknown significance were identified: E152K/H, S153F/L, N155I and E157G. None of the known integrase resistance mutations were observed, except E157Q found in one Cambodian subject (1.1%, CI 95% 0.02-6.3%). The clinical impact of this substitution on resistance of B and nonB-viruses to the licensed INI raltegravir is unclear. If this substitution is confirmed to compromise the virologic response to raltegravir, further studies will be needed to better assess the prevalence of this substitution among CRF01_AE virus.

Published
2011

13. Phylogenetic relationships within Echinococcus and Taenia tapeworms (Cestoda: Taeniidae): an inference from nuclear protein-coding genes

Author

Jenny Knapp, Akira Ito, Minoru Nakao, Antti Lavikainen, Tetsuya Yanagida, Urmas Saarma, Munehiro Okamoto, Laboratoire Chrono-environnement ( LCE ), Université Bourgogne Franche-Comté ( UBFC ) -Centre National de la Recherche Scientifique ( CNRS ) -Université de Franche-Comté ( UFC ), Asahikawa Medical University, Primate Research Institute, Kyoto University [Kyoto], Laboratoire Chrono-environnement - CNRS - UBFC (UMR 6249) (LCE), Centre National de la Recherche Scientifique (CNRS)-Université de Franche-Comté (UFC), and Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université Bourgogne Franche-Comté [COMUE] (UBFC)

Subjects
Paraphyly, MESH: Sequence Analysis, DNA, Time Factors, MESH : Polymorphism, Genetic, MESH: Echinococcus, Helminth genetics, MESH: Base Sequence, MESH: DNA, Helminth, MESH: Genetic Markers, Polymerase Chain Reaction, 030308 mycology & parasitology, MESH : Taenia, MESH : DNA, Helminth, MESH : Genes, Helminth, MESH : Echinococcus, MESH : Genetic Markers, MESH: Animals, MESH: Phylogeny, Clade, MESH : Polymerase Chain Reaction, Genes, Helminth, Phylogeny, 0303 health sciences, Likelihood Functions, Phylogenetic tree, biology, Nuclear Proteins, DNA, Helminth, MESH: Taenia, MESH : RNA Polymerase II, RNA Polymerase II, MESH : Likelihood Functions, MESH : Time Factors, Genetic Markers, MESH: Bayes Theorem, Zoology, MESH : Open Reading Frames, [ SDV.EE.SANT ] Life Sciences [q-bio]/Ecology, environment/Health, 03 medical and health sciences, Open Reading Frames, MESH : Bayes Theorem, Phylogenetics, MESH: Polymorphism, Genetic, parasitic diseases, Genetics, Animals, Humans, Molecular Biology, Ecology, Evolution, Behavior and Systematics, 030304 developmental biology, [SDV.EE.SANT]Life Sciences [q-bio]/Ecology, environment/Health, MESH: Humans, Polymorphism, Genetic, Base Sequence, Taenia, MESH: Time Factors, MESH : Humans, MESH : Phylogeny, MESH : Nuclear Proteins, MESH: Polymerase Chain Reaction, Bayes Theorem, Sequence Analysis, DNA, MESH: Open Reading Frames, biology.organism_classification, MESH: RNA Polymerase II, Echinococcus, Taeniidae, MESH: Likelihood Functions, MESH : Base Sequence, MESH : Animals, MESH: Nuclear Proteins, MESH: Genes, Helminth, MESH : Sequence Analysis, DNA
Abstract

International audience; The family Taeniidae of tapeworms is composed of two genera, Echinococcus and Taenia, which obligately parasitize mammals including humans. Inferring phylogeny via molecular markers is the only way to trace back their evolutionary histories. However, molecular dating approaches are lacking so far. Here we established new markers from nuclear protein-coding genes for RNA polymerase II second largest subunit (rpb2), phosphoenolpyruvate carboxykinase (pepck) and DNA polymerase delta (pold). Bayesian inference and maximum likelihood analyses of the concatenated gene sequences allowed us to reconstruct phylogenetic trees for taeniid parasites. The tree topologies clearly demonstrated that Taenia is paraphyletic and that the clade of Echinococcus oligarthrus and Echinococcusvogeli is sister to all other members of Echinococcus. Both species are endemic in Central and South America, and their definitive hosts originated from carnivores that immigrated from North America after the formation of the Panamanian land bridge about 3 million years ago (Ma). A time-calibrated phylogeny was estimated by a Bayesian relaxed-clock method based on the assumption that the most recent common ancestor of E. oligarthrus and E. vogeli existed during the late Pliocene (3.0 Ma). The results suggest that a clade of Taenia including human-pathogenic species diversified primarily in the late Miocene (11.2 Ma), whereas Echinococcus started to diversify later, in the end of the Miocene (5.8 Ma). Close genetic relationships among the members of Echinococcus imply that the genus is a young group in which speciation and global radiation occurred rapidly.

Published
2010

14. In vivo and ex vivo gene transfer in thymocytes and thymocyte precursors

Author

Adjali , Oumeya, Montel-Hagen , Amélie, Swainson , Louise, Marty , Sophie, Vicente , Rita, Mongellaz , Cedric, Jacquet , Chantal, Zimmermann , Valérie, Taylor , Naomi, Institut de Génétique Moléculaire de Montpellier (IGMM), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Institut de Génétique Moléculaire de Montpellier ( IGMM ), and Université de Montpellier ( UM ) -Centre National de la Recherche Scientifique ( CNRS )

Subjects
MESH : Cell Line, MESH: DNA Primers, MESH: Gene Transfer Techniques, Antigens, CD34, MESH : Transduction, Genetic, Thymus Gland, MESH: Base Sequence, Cell Line, Mice, Transduction, Genetic, MESH : Mice, Animals, Humans, MESH : Thymus Gland, MESH: Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, MESH : Gene Transfer Techniques, [ SDV.BBM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology, MESH: Mice, DNA Primers, MESH: Humans, Base Sequence, MESH : Humans, Gene Transfer Techniques, MESH: Antigens, CD34, MESH: Thymus Gland, MESH: Transduction, Genetic, MESH: Cell Line, MESH : Antigens, CD34, MESH : Base Sequence, MESH : DNA Primers, MESH : Animals
Abstract

International audience; The thymus provides a specialized environment allowing the differentiation of T lymphocytes from bone marrow-derived progenitor cells. We and others have demonstrated that gene transfer into distinct thymocyte populations can be obtained, both in vivo and ex vivo, using lentiviral vectors. Here, we describe techniques for intrathymic lentiviral transduction in mice, using a surgical approach wherein the thoracic cavity is exposed as well as a significantly less invasive strategy wherein virions are directly injected through the skin. Moreover, thymocyte differentiation from murine and human progenitors is now feasible in vitro, under conditions wherein the Notch and IL-7 signaling pathways are activated. We describe methods allowing transduction of murine and human progenitors and their subsequent differentiation into more mature thymocytes. Conditions for lentiviral gene transfer into more differentiated human thymocyte subsets are also presented. Optimization of technologies for HIV-based gene transfer into murine and human thymocyte progenitors will advance strategies aimed at modulating T-cell differentiation and function in-vivo; approaches potentially targeting patients with genetic and acquired immunodeficiencies as well as immune-sensitive tumors. Furthermore, this technology will foster the progression of basic research aimed at elucidating molecular aspects of T-cell differentiation in mice and humans.

Published
2008

15. Epidemiological surveillance of mycoplasmas belonging to the 'Mycoplasma mycoides' cluster: is DGGE fingerprinting of 16S rRNA genes suitable?

Author

Laure Maigre, François Poumarat, A. Tricot, Florence Tardy, Patrice Gaurivaud, Laboratoire d'études et de recherches en pathologie bovine et hygiène des viandes, AFSSA, Mycoplasmoses des ruminants [Lyon], Université de Lyon-VetAgro Sup ( VAS ), Agence Française de Sécurité Sanitaire des Aliments (AFSSA), Mycoplasmoses des Ruminants - UMR (MYCO), and Université de Lyon-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)

Subjects
MESH : Mycoplasma Infections, MESH : Molecular Sequence Data, 16SrRNA gene, MESH: Base Sequence, [ SDV.MP.BAC ] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, medicine.disease_cause, Applied Microbiology and Biotechnology, strains detection and identification, MESH: Mycoplasma, Mycoplasma, MESH : Cattle, RNA, Ribosomal, 16S, MESH: Cattle Diseases, MESH: DNA Fingerprinting, MESH : DNA, Ribosomal, MESH : DNA, Bacterial, MESH: Animals, 1UMYCOPLASME, DGGE, MESH: Phylogeny, Phylogeny, MESH: DNA, Ribosomal, biology, Phylogenetic tree, MESH : Sequence Alignment, Goats, epidemiological surveillance, [ SDV.SPEE ] Life Sciences [q-bio]/Santé publique et épidémiologie, MESH : DNA Fingerprinting, MESH: RNA, Ribosomal, 16S, MESH: Cattle, "Mycoplasma mycoides" cluster, Electrophoresis, Polyacrylamide Gel, Mycoplasma mycoides, Temperature gradient gel electrophoresis, DNA, Bacterial, Sequence analysis, Molecular Sequence Data, MESH: Sequence Alignment, Cattle Diseases, MESH : Goat Diseases, MESH: Goat Diseases, MESH : Electrophoresis, Polyacrylamide Gel, MESH: Goats, DNA, Ribosomal, MYCOPLASME, Microbiology, Phylogenetics, medicine, Animals, Humans, Mycoplasma Infections, MESH : Cattle Diseases, MESH: Humans, MESH: Molecular Sequence Data, MESH : Mycoplasma, Goat Diseases, Base Sequence, MESH : Humans, MESH : Phylogeny, Ribosomal RNA, MESH: Mycoplasma Infections, biology.organism_classification, 16S ribosomal RNA, MESH: DNA, Bacterial, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, DNA Fingerprinting, MESH : RNA, Ribosomal, 16S, MESH : Goats, [SDV.SPEE]Life Sciences [q-bio]/Santé publique et épidémiologie, Mycoplasma mycoides" cluster, MESH : Base Sequence, Cattle, MESH : Animals, Sequence Alignment, MESH: Electrophoresis, Polyacrylamide Gel
Abstract

Aims: The analysis by Denaturing Gradient Gel Electrophoresis (DGGE) of the PCR-amplified V3 region of 16S rRNA gene was previously shown to detect and differentiate a large number of human and animal mycoplasmas. In this study, we further assessed the suitability of the technique for epidemiological surveillance of mycoplasmas belonging to the ‘Mycoplasma mycoides’ cluster, a phylogenetic group that includes major ruminant pathogens. Methods and Results: The V3 region of 16S rRNA genes from approx. 50 field strains was amplified and analysed by DGGE. Detection and identification results were compared with the ones obtained by antigenic testing and sequence analysis. Conclusions: The DGGE technique is robust and valuable as a first-line test, but the patterns obtained for strains belonging to the ‘M. mycoides’ cluster were too variable within a taxon and in contrast too conserved between taxa to allow an unequivocal identification of isolates without further analysis. Significance and Impact of the Study: Issues raised by the quest for a single universal test able to detect and identify any mycoplasma in one clinical sample are thoroughly documented.

Published
2008

16. Tetrameric and homodimeric camelid IgGs originate from the same IgH locus

Author

Pierre Lafaye, Patricia Cavelier, Ikbel Achour, François Rougeon, Christiane Bouchier, Magali Tichit, Génétique et Biochimie du Développement, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique ( CNRS ), Institut de Génétique Moléculaire de Montpellier ( IGMM ), Université de Montpellier ( UM ) -Centre National de la Recherche Scientifique ( CNRS ), Génomique (Plate-Forme), Institut Pasteur [Paris], Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut de Génétique Moléculaire de Montpellier (IGMM), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Génomique (Plate-Forme) - Genomics Platform, Institut Pasteur [Paris] (IP), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)

Subjects
MESH: Immunoglobulins, MESH : Molecular Sequence Data, MESH: Amino Acid Sequence, MESH: Base Sequence, MESH : Immunoglobulins, 0302 clinical medicine, Immunology and Allergy, MESH: Animals, MESH: Phylogeny, Peptide sequence, Phylogeny, Genetics, 0303 health sciences, B-Lymphocytes, biology, MESH : Amino Acid Sequence, MESH: Camels, MESH : Protein Binding, Translocon, medicine.anatomical_structure, MESH : Dimerization, Cosmid, MESH: Camelids, New World, Antibody, Camelids, New World, Dimerization, Protein Binding, Camelus, MESH : Cell Membrane, Immunology, Molecular Sequence Data, Immunoglobulins, [ SDV.IMM.IMM ] Life Sciences [q-bio]/Immunology/Immunotherapy, MESH : B-Lymphocytes, 03 medical and health sciences, Complementary DNA, MESH: B-Lymphocytes, medicine, MESH: Protein Binding, Animals, Amino Acid Sequence, Gene, B cell, 030304 developmental biology, MESH: Molecular Sequence Data, MESH : Camels, Base Sequence, Cell Membrane, MESH : Phylogeny, [SDV.IMM.IMM]Life Sciences [q-bio]/Immunology/Immunotherapy, Molecular biology, MESH : Camelids, New World, Chromosome 4, MESH: Dimerization, biology.protein, MESH : Base Sequence, MESH : Animals, 030215 immunology, MESH: Cell Membrane
Abstract

In addition to producing conventional tetrameric IgGs, camelids have the particularity of producing a functional homodimeric IgG type lacking L (light) chains and only made up of two H (heavy) chains. This nonconventional IgG type is characterized by variable and constant regions referred to as VHH and CHH, respectively, and which differ from conventional VH and CH counterparts. Although the structural properties of homodimeric IgGs have been well investigated, the genetic bases involved in their generation are still largely unknown. In this study, we characterized the organization of genes coding for the H chains of tetrameric and homodimeric IgGs by constructing an alpaca (Lama pacos) genomic cosmid library. We showed that a single IgH locus in alpaca chromosome 4 contains all of the genetic elements required for the generation of the two types of Igs. The alpaca IgH locus is composed of a V region that contains both VHH and VH genes followed by a unique DH-JH cluster and C region genes, which include both CHH and CH genes. Although this general gene organization greatly resembles that of other typical mammalian Vn-Dn-Jn-Cn translocon IgH loci, the intermixed gene organization within the alpaca V and C regions reveals a new type of translocon IgH locus. Furthermore, analyses of cDNA coding for the membrane forms of IgG and IgM present in alpaca peripheral blood B cells are most consistent with the notion that the development of a B cell bearing homodimeric IgG passes through an IgM+ stage, similar to the case for conventional IgG.

Published
2008

17. Persistent Wolbachia and Cultivable Bacteria Infection in the Reproductive and Somatic Tissues of the Mosquito Vector Aedes albopictus

Author

Karima Zouache, Laurence Mousson, Van Tran-Van, Denis Voronin, Anna-Bella Failloux, Patrick Mavingui, Laboratoire d'Ecologie Microbienne - UMR 5557 (LEM), Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Ecole Nationale Vétérinaire de Lyon (ENVL), Génétique Moléculaire des Bunyavirus, Institut Pasteur [Paris], KZ was supported by a PhD fellowship from the French Ministère de l'Education Nationale, de la Recherche et des Nouvelles Technologies. This work was funded by the CNRS and the Agence Nationale de Recherche (ChikVendoM ANR-06-SEST07). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., ANR-06-SEST-0007,CHIKVENDOM,Interactions multipartites entre le virus chikungunya, les endosymbiostes et les moustiques. Impact sur la transmission virale et la dynamique des populations vectorielles(2006), Ecologie microbienne ( EM ), Centre National de la Recherche Scientifique ( CNRS ) -Ecole Nationale Vétérinaire de Lyon ( ENVL ) -Université Claude Bernard Lyon 1 ( UCBL ), Université de Lyon-Université de Lyon-Institut National de la Recherche Agronomique ( INRA ) -VetAgro Sup ( VAS ), ANR-06-SEST-0007,CHIKVENDOM,Interactions multipartites entre le virus chikungunya, les endosymbiostes et les moustiques. Impact sur la transmission virale et la dynamique des populations vectorielles ( 2006 ), Centre National de la Recherche Scientifique (CNRS)-Ecole Nationale Vétérinaire de Lyon (ENVL)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Recherche Agronomique (INRA)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS), Université de Lyon-Université de Lyon-Ecole Nationale Vétérinaire de Lyon (ENVL)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris] (IP)

Subjects
viruses, lcsh:Medicine, MESH: Base Sequence, MESH: Reproduction, medicine.disease_cause, Polymerase Chain Reaction, law.invention, Dengue fever, Aedes, law, MESH : Insect Vectors, MESH : Female, MESH: Animals, MESH: In Situ Hybridization, Fluorescence, Chikungunya, lcsh:Science, MESH : Polymerase Chain Reaction, In Situ Hybridization, Fluorescence, Polymerase chain reaction, 0303 health sciences, Multidisciplinary, Reproduction, virus diseases, MESH : Reproduction, MESH: Aedes, Genitalia, Female, 3. Good health, MESH : In Situ Hybridization, Fluorescence, MESH : Genitalia, Female, MESH : Wolbachia, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, MESH: Wolbachia, MESH : DNA Primers, Female, Wolbachia, Microbiology/Cellular Microbiology and Pathogenesis, Research Article, MESH: DNA Primers, Aedes albopictus, MESH: Insect Vectors, Biology, [ SDV.MP.VIR ] Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Microbiology, 03 medical and health sciences, parasitic diseases, medicine, Animals, Microbiology/Environmental Microbiology, MESH : Aedes, DNA Primers, 030304 developmental biology, Base Sequence, 030306 microbiology, Host (biology), lcsh:R, MESH: Polymerase Chain Reaction, biochemical phenomena, metabolism, and nutrition, biology.organism_classification, medicine.disease, Virology, Insect Vectors, Vector (epidemiology), lcsh:Q, MESH : Base Sequence, MESH : Animals, MESH: Genitalia, Female, MESH: Female, Bacteria
Abstract

International audience; BACKGROUND:Commensal and symbiotic microbes have a considerable impact on the behavior of many arthropod hosts, including hematophagous species that transmit pathogens causing infectious diseases to human and animals. Little is known about the bacteria associated with mosquitoes other than the vectorized pathogens. This study investigated Wolbachia and cultivable bacteria that persist through generations in Ae. albopictus organs known to host transmitted arboviruses, such as dengue and chikungunya.METHODOLOGY/PRINCIPAL FINDINGS:We used culturing, diagnostic and quantitative PCR, as well as in situ hybridization, to detect and locate bacteria in whole individual mosquitoes and in dissected tissues. Wolbachia, cultivable bacteria of the genera Acinetobacter, Comamonas, Delftia and Pseudomonas co-occurred and persisted in the bodies of both males and females of Ae. albopictus initially collected in La Réunion during the chikungunya outbreak, and maintained as colonies in insectaries. In dissected tissues, Wolbachia and the cultivable Acinetobacter can be detected in the salivary glands. The other bacteria are commonly found in the gut. Quantitative PCR estimates suggest that Wolbachia densities are highest in ovaries, lower than those of Acinetobacter in the gut, and approximately equal to those of Acinetobacter in the salivary glands. Hybridization using specific fluorescent probes successfully localized Wolbachia in all germ cells, including the oocytes, and in the salivary glands, whereas the Acinetobacter hybridizing signal was mostly located in the foregut and in the anterior midgut.CONCLUSIONS/SIGNIFICANCE:Our results show that Proteobacteria are distributed in the somatic and reproductive tissues of mosquito where transmissible pathogens reside and replicate. This location may portend the coexistence of symbionts and pathogens, and thus the possibility that competition or cooperation phenomena may occur in the mosquito vector Ae. albopictus. Improved understanding of the vectorial system, including the role of bacteria in the vector's biology and competence, could have major implications for understanding viral emergences and for disease control.

Published
2009

18. Evaluation of acyl coenzyme A oxidase (Aox) isozyme function in the n- alkane-assimilating yeast Yarrowia lipolytica

Author

Jean-Marc Belin, Céline Laroche, Jean-Marc Nicaud, Huijie J. Wang, Yves Waché, Marie-Thérèse Le Dall, Claude Gaillardin, Microbiologie et Génétique Moléculaire (MGM), Institut National de la Recherche Agronomique (INRA)-Institut National Agronomique Paris-Grignon (INA P-G)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Microbioologie, Institut National de la Recherche Agronomique (INRA)-Université de Bourgogne (UB)-Ecole Nationale Supérieure de Biologie Appliquée à la Nutrition et à l'Alimentation de Dijon (ENSBANA), Génie des Procédés Microbiologiques et Alimentaires (GPMA), Université de Bourgogne (UB)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement, Collection de Levures d'Intérêt Biotechnologique et Laboratoire de Génétique Moléculaire et Cellulaire, Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Wache, Yves, Microbiologie et Génétique Moléculaire ( MGM ), Institut National de la Recherche Agronomique ( INRA ) -Institut National Agronomique Paris-Grignon ( INA P-G ) -Centre National de la Recherche Scientifique ( CNRS ), Laboratoire de Microbioologie, UMR INRA 1283 Ecole Nationale Supérieure de Biologie Appliquée à la. Nutrition et à l'Alimentation, Institut National de la Recherche Agronomique ( INRA ), Génie des Procédés Microbiologiques et Alimentaires ( GPMA ), Université de Bourgogne ( UB ) -AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement, and Institut National de la Recherche Agronomique ( INRA ) -Centre National de la Recherche Scientifique ( CNRS ) -Centre National de la Recherche Scientifique ( CNRS )

Subjects
MESH : Escherichia coli, MESH: Sequence Analysis, DNA, MESH : Molecular Sequence Data, Mutant, Gene Expression, MESH: Base Sequence, chemistry.chemical_compound, Cloning, Molecular, [INFO.INFO-BT]Computer Science [cs]/Biotechnology, DNA, Fungal, MESH: Mutagenesis, MESH : Isoenzymes, Oxidase test, biology, MESH: Escherichia coli, MESH: Acyl-CoA Oxidase, MESH : Mutagenesis, MESH : Cell Division, MESH : Oxidoreductases, Isoenzymes, Blot, Eukaryotic Cells, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Fungal, Biochemistry, MESH: Isoenzymes, MESH: Cell Division, MESH : Acyl-CoA Oxidase, Oxidoreductases, Sequence Analysis, [ INFO.INFO-BT ] Computer Science [cs]/Biotechnology, Cell Division, MESH: Gene Expression, MESH : Cloning, Molecular, Genes, Fungal, Molecular Sequence Data, Microbiology, Isozyme, WESTERN BLOTTING, Alkanes, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Escherichia coli, MESH: Cloning, Molecular, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, MESH: Oxidoreductases, MESH: Saccharomycetales, [ SDV.BBM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, Gene, MESH : Alkanes, MESH: Molecular Sequence Data, Base Sequence, Molecular, Yarrowia, Sequence Analysis, DNA, MESH : Saccharomycetales, DNA, biology.organism_classification, Molecular biology, Yeast, MESH : Gene Expression, MESH: Alkanes, MESH: DNA, Fungal, Oleic acid, [INFO.INFO-BT] Computer Science [cs]/Biotechnology, Genes, chemistry, Mutagenesis, Saccharomycetales, MESH : Base Sequence, MESH : Genes, Fungal, Acyl-CoA Oxidase, MESH : DNA, Fungal, MESH: Genes, Fungal, MESH : Sequence Analysis, DNA, Cloning
Abstract

We have identified five acyl coenzyme A (CoA) oxidase isozymes (Aox1 through Aox5) in the n -alkane-assimilating yeast Yarrowia lipolytica , encoded by the POX1 through POX5 genes. The physiological function of these oxidases has been investigated by gene disruption. Single, double, triple, and quadruple disruptants were constructed. Global Aox activity was determined as a function of time after induction and of substrate chain length. Single null mutations did not affect growth but affected the chain length preference of acyl-CoA oxidase activity, as evidenced by a chain length specificity for Aox2 and Aox3. Aox2 was shown to be a long-chain acyl-CoA oxidase and Aox3 was found to be active against short-chain fatty acids, whereas Aox5 was active against molecules of all chain lengths. Mutations in Aox4 and Aox5 resulted in an increase in total Aox activity. The growth of mutant strains was analyzed. In the presence of POX1 only, strains did not grow on fatty acids, whereas POX4 alone elicited partial growth, and the growth of the double POX2-POX3 -deleted mutant was normal excepted on plates containing oleic acid as the carbon source. The amounts of Aox protein detected by Western blotting paralleled the Aox activity levels, demonstrating the regulation of Aox in cells according to the POX genotype.

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